CN110320204A - The colorimetric detection method of yapamicin relict in a kind of milk - Google Patents
The colorimetric detection method of yapamicin relict in a kind of milk Download PDFInfo
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- CN110320204A CN110320204A CN201810296903.7A CN201810296903A CN110320204A CN 110320204 A CN110320204 A CN 110320204A CN 201810296903 A CN201810296903 A CN 201810296903A CN 110320204 A CN110320204 A CN 110320204A
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- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 239000008267 milk Substances 0.000 title claims abstract description 23
- 210000004080 milk Anatomy 0.000 title claims abstract description 23
- 235000013336 milk Nutrition 0.000 title claims abstract description 23
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims abstract description 54
- 229930027917 kanamycin Natural products 0.000 claims abstract description 54
- 239000000523 sample Substances 0.000 claims abstract description 23
- 238000012986 modification Methods 0.000 claims abstract description 21
- 239000013256 coordination polymer Substances 0.000 claims abstract description 17
- 239000011324 bead Substances 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 16
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000010931 gold Substances 0.000 claims abstract description 9
- 229910052737 gold Inorganic materials 0.000 claims abstract description 9
- 108091023037 Aptamer Proteins 0.000 claims abstract description 5
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 5
- 229960002685 biotin Drugs 0.000 claims abstract description 5
- 235000020958 biotin Nutrition 0.000 claims abstract description 5
- 239000011616 biotin Substances 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims abstract description 4
- 102000053602 DNA Human genes 0.000 claims abstract description 4
- 230000008836 DNA modification Effects 0.000 claims abstract description 4
- 238000009396 hybridization Methods 0.000 claims abstract description 4
- 238000002372 labelling Methods 0.000 claims abstract description 4
- 230000035945 sensitivity Effects 0.000 claims abstract description 3
- 238000011534 incubation Methods 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 230000004048 modification Effects 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000002835 absorbance Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000011896 sensitive detection Methods 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims 1
- 239000001913 cellulose Substances 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- 239000000243 solution Substances 0.000 description 9
- 230000003321 amplification Effects 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000007981 phosphate-citrate buffer Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010002199 Anaphylactic shock Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229960005475 antiinfective agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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Abstract
The colorimetric detection method of yapamicin relict, belongs to technical field of analytical chemistry in a kind of milk.The present invention is first by kanamycins aptamer (APTKNA) with the probe CP of biotin labelingKNAAnneal forms double-stranded DNA modification in the surface Streptavidin coupled bead (SDB), probe SPKNAAnd HPKNAModification is on the surface gold nano grain (AuNPs)；When in system there are when kanamycins (KNA), KNA and APTKNASpecific binding and make APTKNAIt is detached from SDB, and then CPKNAThe HP then modified with AuNPsKNAHybridization forms SDB-AuNPs system；The SP of AuNPs surface modificationKNAIn conjunction with OPD-H can be catalyzed after SAv-HRP2O2Solution changes color draws standard curve using the variation of ultraviolet absorptivity at 450nm and the relationship of kanamycins concentration, and by measuring the ultraviolet absorptivity of sample to be tested, the detection of kanamycins concentration can be realized.This method have high sensitivity, low cost, it is easy to operate the features such as, it can be achieved that in sample kanamycins sensitive determination.
Description
Technical field
The present invention is a kind of colorimetric detection method of yapamicin relict in milk, especially yapamicin relict in milk
Detection method, belong to analytical chemistry field.
Background technique
Kanamycins is a kind of aminoglycoside for being effectively used for Gram-negative bacteria and gram positive bacteria infection treatment
Class broad-spectrum antibiotic is clinically the most commonly used one of anti-infectives, extensive since its active force is relatively strong and lasting
Treatment for the mankind and the forms such as animal injection, oral.However kanamycins or long-term consumption is excessively used containing card
The remaining animal derived food of that mycin then will lead to a series of adverse reactions, including ear, Toxicity of Kidney and anaphylactic shock
Deng.Therefore, the detection method for establishing food source antibiotic residue is of great significance to actual production life.
Gold nano grain is widely used in one of the nano material of signal amplification because of its unique physics and chemical property.
Other many functional moleculars can be coupled on its surface, while successfully keeping its original biological nature, possess better stability.
Beads enrichment technology and other isolation technics (such as chromatographic isolation, centrifuge separation and UF membrane), which are compared, has high throughput, low cost,
Significant advantage that is quick and simplifying operation.
At present detection kanamycins common technique mainly include there is several methods that, such as enzyme-linked immunosorbent assay, capillary
Electrophoresis tube, high performance liquid chromatography, surface plasma body resonant vibration, electrochemical method and colorimetric method.It is most of in these methods
There is certain defect, cumbersome sample preprocessing program, the expensive reagents of immuno-chemical method, detection time are long, complexity
Instrument.Therefore, by Beads enrichment and the amplification of gold nano grain signal can establish it is a kind of it is sensitive efficiently, it is cheap, easy to be fast
The detection method of prompt kanamycins is particularly important.
Summary of the invention
The purpose of the present invention is combining Beads enrichment with the advantage that the signal of gold nano grain amplifies, one kind is established
It is sensitive efficient, the detection method of cheap, simple and efficient kanamycins.
Technical solution of the present invention: the present invention is a kind of colorimetric detection method of yapamicin relict in milk, first will
Kanamycins aptamer (APTKNA) with the probe CP of biotin labelingKNAAnneal forms double-stranded DNA modification in Streptavidin
The surface coupled bead (SDB)；Probe SPKNAAnd HPKNAModification is on the surface gold nano grain (AuNPs).Block that when existing in system
When mycin (KNA), KNA and APTKNASpecific binding and make APTKNAIt is detached from SDB, and then CPKNAThen modified with AuNPs
HPKNAHybridization forms SDB-AuNPs system；The SP of AuNPs surface modificationKNAIn conjunction with OPD-H can be catalyzed after SAv-HRP2O2Solution becomes
Color draws standard curve using the variation of ultraviolet absorptivity at 450nm and the relationship of kanamycins concentration, to be measured by measuring
The detection of kanamycins concentration can be realized in the ultraviolet absorptivity of sample.
Method is the following steps are included: the pretreatment of magnetic bead, the preparation of probe modification SDB, the preparation of gold nano grain, probe
Modify preparation, the sample incubation of AuNPs, UV spectrophotometer measuring.
(1) pretreatment of magnetic bead
Magnetic bead (SDB) stoste for taking the Streptavidin of 10mg/mL to be coupled is added in EP pipe and contains 1mM EDTA, 2M
The 10mM Tris-HCl (pH 7.5) of NaCl is rinsed magnetic bead 3 times, and SDB finally is resuspended with 10mM PBS (pH 7.0), obtains 2mg/
ML is clean, and magnetic bead is spare.
(2) preparation of probe modification SDB
By 20 μ L, 10 μM of APTKNAWith 20 μ L, 10 μM of CPKNAIt is added to 210 μ L NaCl containing 0.3M, 10mM PBS (pH
7.0) in solution, the mixed liquor of 250 μ L of total volume is obtained, which is slowly dropped to room temperature after 90 DEG C of heating 2min.?
The mixed liquor of 250 μ L is added pretreated 50 μ L magnetic bead solution in above-mentioned (1) and mixes, in 37 DEG C of incubation 90min, with 10mM PBS
After (pH 7.0) is washed 3 times, after 500 μ L 2%BSA incubation at room temperature 1h is added, by CPKNA/APTKNAThe SDB modified is resuspended in
50 μ L 2mg/mL CP are obtained in 50 μ L 10mM PBS (pH 7.0)KNA/APTKNAThe SDB of modification.
Above-mentioned APTKNASequence are as follows: 5 '-AGA TGG GGG TTG AGG CTA AGC CGA-3 '
Above-mentioned CPKNASequence are as follows: 5 '-Biotin-ATA TAT ATA TTC GGC TTA GCC TCA ACC CCC ATC T-
3′
(3) preparation of AuNPs
All glasswares needed for preparation must be in newly configuration chloroazotic acid (HNO3: HCl=3: 1) after impregnating 30min in, use
A large amount of distilled water is rinsed and is dried for standby in baking box.The preparation process of gold nano grain is as follows: preparing 100mL0.01%
Gold chloride be added three-necked bottle in, be stirred and heated to boiling.After it is boiled, it is rapidly added 3.5mL with vigorous stirring
1% trisodium citrate, continue heating stirring 15min after to solution become claret close heater, continue stir 30min,
Blender is closed to deposit in the gold nano grain prepared (AuNPs) in brown reagent bottle simultaneously after solution is cooled to room temperature
4 DEG C of refrigerators are placed on to save.
(4) preparation of probe modification AuNPs
By the HP of 10 μM of AuNPs and 4 μ L being prepared in 100 μ L above-mentioned (3)KNA, 10 μM of 40 μ L SPKNAAnd 356 μ L
500 μ L mixed liquors are made in the 10mM PBS that pH value is 7.0.By the mixed liquor after 37 DEG C of incubation 1h, 12000rpm, 4 DEG C of centrifugations
20min after abandoning 485 μ L of supernatant, adds 485 μ L 10mM PBS (pH 7.0) that (repeated centrifugation is washed 3 times) is resuspended, obtains total volume
SP is modified for 500 μ LKNA/HPKNAGold nano grain.
Above-mentioned HPKNASequence are as follows: 5 '-SH-AAA AAA AGA TGG GGG TTG-3 '
Above-mentioned SPKNASequence are as follows: 5 '-Biotin-ATA TAT ATA TTC GGC TTA GCC TCA AAA AA-SH-3 '
(5) sample incubation
By the 2mg/mL surface modification prepared in above-mentioned (2) CPKNA/APTKNA4 μ L of SDB and 4 μ L various concentrations
Mixed liquor is made in the 10mM PBS (pH 7.4) of KNA and 2 μ L NaCl containing 0.1M, by the mixed liquor in 37 DEG C of incubation 1h, uses 10mM
PBS (pH 7.0) is cleaned 3 times, and the surface modification prepared in 12.5 μ L above-mentioned (4) HP is addedKNA/SPKNAGold nano grain in
37 DEG C of incubation 1h.It is cleaned 3 times with 100 μ L 10mM PBS (pH 7.0), is resuspended with 10mM PBS (pH 7.0).
(6) UV spectrophotometer measuring
The SAv-HRP (0.01mg/mL) that 0.5 μ L is added in solution in above-mentioned (5) is incubated for 1h at 37 DEG C, with 200 μ L
10mM PBS (pH 6.0) adds 600 μ L OPD-H after cleaning 5 times2O2(OPD containing 7.4mM and 5.9mM H2O220mM phosphate-
Citrate buffer, pH 5.0) it is resuspended and observes color change after magnetic bead is incubated for 30 minutes in 37 DEG C and detect ultraviolet at 450nm
Absorbance.
The concentration of kanamycins is 10-6To 105When pg/mL, solution colour gradually becomes buff from transparent, in 450nm
The absorbance at place increases with the raising of kanamycins concentration, at 450nm ultraviolet absorptivity and concentration there are good linear dependence,
Meet quantitative requirement.
Beneficial effects of the present invention: 1. this method designs different DNA probes, magnetic bead using kanamycins (KNA) aptamer
Quick separating and AuNPs mediate signal amplification and selectivity organism colorimetric sensor advantage, realize in milk and block that
The detection of mycin, sensitivity is high, has weight to the detection of kanamycins wide scope range of linearity quantification, Sensitive Detection in milk
Want meaning；2. there is universal reference in methodology, by replacing DNA sequence dna, so that it may design Multiple Classes of Antibiotics inspection
Highly sensitive at low cost, easy to operate, the analysis method without complicated procedures of survey.
Detailed description of the invention
Fig. 1 is a kind of colorimetric detection method schematic diagram of yapamicin relict in milk
Fig. 2 is a kind of colorimetric detection method specificity figure of yapamicin relict in milk
Fig. 3 is the concentration relationship figure of ultraviolet absorptivity and kanamycins in the presence of various concentration kanamycins
Specific embodiment
The measurement of the colorimetric detection method specificity of yapamicin relict in a kind of milk of embodiment 1.
Under optimum experimental condition, the verifying of system specificity has been carried out.The CP that will be prepared in above method step (2)KNA/
APTKNAThe different types of antibiotic of 4 μ L of SDB and 4 μ L of modification and the 10mM PBS (pH 7.4) of 2 μ L NaCl containing 0.1M are made
SP in 12.5 μ L above-mentioned steps (4) is added after 37 DEG C of incubation 1h, 10mM PBS (pH 7.0) are cleaned 3 times in mixed liquorKNA/HPKNA
The AuNPs of modification is cleaned and is resuspended with 10mM PBS (pH 7.0) in 37 DEG C of incubation 1h, and the SAv-HRP of 0.5 μ L is added
(0.01mg/mL) is cleaned 5 times in 37 DEG C of incubation 1h with 200 μ L 10mM PBS (pH 6.0), adds 600 μ L OPD-H2O2Magnetic is resuspended
Pearl observes color change after 37 DEG C of incubation 30min and detects ultraviolet absorptivity at 450nm.The scanning result measured such as Fig. 2A,
Shown in 2B, this method has good selectivity to detection kanamycins.
In the presence of 2. various concentration kanamycins of embodiment, the survey of the concentration standard curve of ultraviolet absorptivity and kanamycins
It is fixed
The kanamycins of the 4 μ L SDB and 4 μ L various concentrations that prepare in above method step (2) and 2 μ L are contained into 0.1M
NaCl, the 10mM PBS that pH value is 7.4 are made mixed liquor and are added 12.5 after 37 DEG C of incubation 1h, 10mM PBS (pH 7.0) are cleaned
SP in μ L above-mentioned steps (4)KNA/HPKNAThe AuNPs of modification is cleaned and is resuspended with 10mM PBS (pH 7.0) in 37 DEG C of incubation 1h,
Then the SAv-HRP (0.01mg/mL) that 0.5 μ L is added is incubated for after 1h with 200 μ L 10mM PBS (pH 6.0) clearly at 37 DEG C
It washes, adds 600 μ L OPD-H2O2(OPD containing 7.4mM and 5.9mM H2O220mM phosphate citrate buffer, pH 5.0)
Magnetic bead is resuspended to observe color change after 37 DEG C of incubation 30min and detect the ultraviolet absorptivity at 450nm.The result of detection is as schemed
Shown in 3A-C, when the concentration of KNA is 10-6To 105When pg/mL, solution colour gradually becomes buff from transparent, at 450nm
Absorbance (Abs450nm) with KNA concentration show that good linear relationship, the two meet Abs450nm=0.0850logcKAN+
0.6650(R2=0.997), wherein Abs represents absorbance value, and c represents the concentration of KNA (pg/mL).
The method that normal concentration kanamycins preparation artificial contamination's milk is added into milk obtains the milk of kanamycins
Sample substitutes the kanamycins standard solution of above-mentioned various concentration, shown in experimental result (table 1), it was demonstrated that the system has practical
Operability, good confidence level and repeatability.
1. milk sample measured result of table
Claims (5)
1. the colorimetric detection method of yapamicin relict, belongs to technical field of analytical chemistry in a kind of milk.The present invention first will
Kanamycins aptamer (APTKNA) with the probe CP of biotin labelingKNAAnneal forms double-stranded DNA modification in Streptavidin
The surface coupled bead (SDB), probe SPKNAAnd HPKNAModification is on the surface gold nano grain (AuNPs)；Block that when existing in system
When mycin (KNA), KNA and APTKNASpecific binding and make APTKNAIt is detached from SDB, and then CPKNAThen modified with AuNPs
HPKNAHybridization forms SDB-AuNPs system；The SP of AuNPs surface modificationKNAIn conjunction with OPD-H can be catalyzed after SAv-HRP2O2Solution becomes
Color draws standard curve using the variation of ultraviolet absorptivity at 450nm and the relationship of kanamycins concentration, to be measured by measuring
The detection of kanamycins concentration can be realized in the ultraviolet absorptivity of sample.This method has high sensitivity, and low cost is easy to operate
The features such as, it can be achieved that in sample kanamycins sensitive determination.
2. the colorimetric detection method of yapamicin relict in a kind of milk according to claim 1, it is characterized in that will be blocked
Mycin aptamer (APTKNA) with the probe CP of biotin labelingKNAAnneal forms double-stranded DNA modification and is coupled in Streptavidin
Surface magnetic bead (SDB), by 20 μ L, 10 μM of APTKNAWith 20 μ L, 10 μM of CPKNA210 μ L NaCl containing 0.3M are added to, pH value is
In 7.0 10mM PBS, it is slowly dropped to room temperature after 90 DEG C of heating 2min, 50 μ L SDB, 37 DEG C of incubation 90min are added later, is used
After the 10mM PBS that pH value is 7.0 is washed 3 times, after 500 μ L 2%BSA incubation at room temperature 1h is added, by CPKNA/APTKNAIt modifies
SDB be resuspended in 50 μ L pH value be 7.0 10mM PBS solution in.
3. the colorimetric detection method of yapamicin relict in a kind of milk according to claim 1, it is characterized in that biotin
The probe SP of modificationKNAAnd HPKNABy sulfydryl modification on the surface AuNPs, the 100 μ L of AuNPs of preparation is added 10 μM of 4 μ L's
HPKNA, 10 μM of 40 μ L SPKNAThe 10mM PBS for being 7.0 with 356 μ L pH value is in 37 DEG C of 1h, 12000rpm, 4 DEG C of incubation centrifugations
20min washs 3 times with the 10mM PBS repeated centrifugation that pH value is 7.0 and obtains HPKNA/SPKNAThe AuNPs of modification.
4. the colorimetric detection method of yapamicin relict in a kind of milk according to claim 1, it is characterized in that working as system
In there are when kanamycins (KNA), KNA and APTKNASpecific binding and make APTKNAIt is detached from SDB, and then CPKNAThen with
The HP of AuNPs modificationKNAHybridization forms SDB-AuNPs system, by CP in the claims 2KNA/APTKNA4 μ of SDB of modification
L, the KNA of 4 μ L various concentrations and 2 μ L NaCl containing 0.1M, the 10mM PBS that pH value is 7.4 are made mixed liquor and are incubated in 37 DEG C
HP in 12.5 μ L the claims 3 is added after being washed with the 10mM PBS that pH value is 7.0 in 1hKNA/SPKNAThe AuNPs of modification in
37 DEG C of incubation 1h are cleaned 3 times and are resuspended and formed SDB-AuNPs system with the 10mM PBS that 100 μ L pH value are 7.0.
5. the colorimetric detection method of yapamicin relict in a kind of milk according to claim 1, it is characterized in that utilizing
The relationship of the variation of ultraviolet absorptivity and kanamycins concentration at 450nm formulates standard curve, can be realized that block that in sample mould
The Sensitive Detection of cellulose content.0.5 μ L SAv-HRP will be added in the SDB-AuNPs system formed in the claims 4 in 37
It is incubated for 1h at DEG C, is cleaned 5 times with the 10mM PBS that 200 μ L pH value are 6.0, adds 600 μ L OPD-H2O2Solution is resuspended in 37 DEG C and incubates
It educates 30min observation color change and detects ultraviolet absorptivity at 450nm.Detect the ultraviolet of the kanamycins of series of standards concentration
Absorbance draws standard curve, and manually the method for contaminated milk obtains the above-mentioned series mark of milk sample substitution of kanamycins
The kanamycins solution of quasi- concentration, the dense of kanamycins in practical milk can be calculated by obtaining ultraviolet absorptivity by standard curve
Degree.
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